Vulnerability to psychosocial disability in psychosis.

Psychosocial disability affects a number of individuals with psychosis and often begins years before the formal onset of disorder. This suggests that for many, their psychosocial disability is enduring, and targeted interventions are therefore needed earlier in their developmental trajectories to ensure that psychosocial disability does not become entrenched. Poor psychosocial functioning also affects individuals with a range of different emerging mental health problems, putting these young people at risk of long-term social marginalisation and economic disadvantage; all of which are known risk factors for the development of psychosis. Identification of the markers of poor psychosocial functioning will help to inform effective treatments. This editorial will discern the early trajectories and markers of poor psychosocial outcome in psychosis, and highlight which individuals are most at risk of having a poor outcome. This editorial will also discuss whether early interventions are currently being targeted appropriately and will propose how intervention and preventative strategies can be implemented, to restore psychosocial trajectories in a way that enables young people to maximise their life chances.

Using the internet for rapid investigation of an outbreak of diarrhoeal illness in mountain bikers.

In summer 2008, we investigated an outbreak of diarrhoeal illness in participants of a mountain-bike event in Wales (UK) which had been affected by heavy rain. We conducted a retrospective cohort study to investigate the cause using an internet-based questionnaire. Fifty-three percent of those contacted responded, and 161 (46·5%) out of the 347 responders, reported gastrointestinal symptoms. Median day of onset was 3 days following the event. Ten riders reported receiving a laboratory-confirmed diagnosis of Campylobacter. Multivariate logistic regression analysis identified the inadvertent ingestion of mud (OR 2·5, 95% CI 1·5-4·2, P<0·001) and eating 'other' food during the event (OR 2·1, 95% CI 1·2-3·6, P=0·01) as significant risk factors for illness. We concluded that the outbreak was caused by Campylobacter, spread to the riders by the inadvertent ingestion of mud which had been contaminated with sheep faeces from the rural course. Mountain-bike race organizers should consider microbiological hazards when risk-assessing potential race courses. The internet is an efficient tool for the investigation of outbreaks in computer-literate populations.

The effects of N-ras oncogene expression on PDGF-BB stimulated responses in cultured mouse myoblasts.

The role of the ras oncogene in the signalling pathway triggered by platelet-derived growth factor BB (PDGF-BB) has been investigated in a cell line which normally differentiates into myotubes. Following the activation of the N-ras oncogene, however, the cells proliferate and form foci. PDGF-BB stimulated the phosphorylation of tyrosine in several cellular proteins of molecular weight 185, 160, 94, 54, 44, 42 kDa and furthermore Ca2+ was released from internal stores. Activation of the N-ras gene by treatment of cells with dexamethasone (DEX) inhibited these responses to PDGF-BB. On the other hand, both ras-induced and -non induced cells responded to bradykinin (BK), foetal calf serum (FCS) and ionomycin (ION) by releasing Ca2+ from intracellular stores. The inhibition of the response to PDGF-BB in ras-activated cells has been further investigated. The binding of [125I]-PDGF-BB to its receptors was low and western blotting showed a low level of PDGF-BB receptor protein. This was in marked contrast to the receptor number seen in cells grown in growth medium or fusion promoting medium. These results indicate that cells transformed with the N-ras oncogene fail to respond to platelet-derived growth factor and exhibit a very low level of PDGF receptors. This suggests a role for the ras oncogene in the earliest steps of the signalling pathway.

Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum.

An antiserum was generated against a dodecapeptide whose sequence is found at the C-terminus of a cyclic AMP (cAMP)-specific, type-IVA phosphodiesterase encoded by the rat 'dunc-like' cyclic AMP phosphodiesterase (RD1) cDNA. This antiserum identified a single approximately 73 kDa protein species upon immunoblotting of cerebellum homogenates. This species co-migrated upon SDS/PAGE with a single immunoreactive species observed in COS cells transfected with the cDNA for RD1. Native RD1 in cerebellum was found to be predominantly (approximately 93%) membrane-associated and could be found in isolated synaptosome populations, in particular those enriched in post-synaptic densities. Fractionation of lysed synaptosomes on sucrose density gradients identified RD1 as co-migrating with the plasma membrane marker 5'-nucleotidase. Laser scanning confocal and digital deconvolution immunofluorescence studies done on intact COS cells transfected with RD1 cDNA showed RD1 to be predominantly localized to plasma membranes but also associated with the Golgi apparatus and intracellular vesicles. RD1-specific antisera immunoprecipitated phosphodiesterase activity from solubilized cerebellum membranes. This activity had the characteristics expected of the type-IV cAMP phosphodiesterase RD1 in that it was cAMP specific, exhibited a low Km cAMP of 2.3 microM, high sensitivity to inhibition by 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) (Ki approximately 0.7 microM) and was unaffected by Ca2+/calmodulin and low concentrations of cyclic GMP. The phosphodiesterase activities of RD1 solubilized from both cerebellum and transfected COS cell membranes showed identical first-order thermal denaturation kinetics at 50 degrees C. Native RD1 from cerebellum was shown to be an integral protein in that it was solubilized using the non-ionic detergent Triton X-100 but not by either re-homogenization or high NaCl concentrations. The observation that hydroxylamine was unable to cause the release of RD1 from either cerebellum or COS membranes and that [3H]palmitate was not incorporated into the RD1 protein immunoprecipitated from COS cells transfected with RD1 cDNA, indicated that RD1 was not anchored by N-terminal acylation. The engineered deletion of the 25 residues forming the unique N-terminal domain of RD1 caused both a profound increase in its activity (approximately 2-fold increase in Vmax) and a profound change in intracellular distribution. Thus, immunofluorescence studies identified the N-terminal truncated species as occurring exclusively ion the cytosol of transfected COS cells. The cDNA for RD1 thus appears to encode a native full-length type-IVA phosphodiesterase that is expressed in cerebellum.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterisation of the binding sites for Escherichia coli heat-labile toxin type I in intestinal brush borders.

Intestinal brush borders from Wistar rats contained a total of 20-30-times more binding sites for Escherichia coli heat-labile enterotoxin (LT-1) than for cholera toxin (CT). The results suggest that LT-1 binds to sites in addition to ganglioside GM1, the binding site for CT. Brush border proteins were separated by SDS-PAGE, blotted to nitrocellulose and the filters incubated with 125I-labeled toxins. [125I]LT-1 was shown to bind to a series of brush border galactoproteins ranging in size from 130-140 kDa. Binding was inhibited by unlabeled LT-1 (but not CT), and by ricin and free galactose. A number of brush border enzymes are large glycoproteins which can be solubilised by papain. The papain-solubilised sucrase-isomaltase complex was purified by affinity chromatography and shown to bind LT-1, as did the proteins in fractions enriched in maltase activity. However, such brush border galactoproteins do not account for all of the additional LT-1 binding sites. Thus, brush borders prepared from 1-15-day-old rabbits contained many more binding sites for LT-1 than CT despite the absence of any sucrase-isomaltase activity, and no [125I]LT-1 binding proteins could be detected by blotting. There was a marked variation in the number of LT-1 binding sites in different strains of rat, and between different species.

Diabetes-induced changes in guanine-nucleotide-regulatory-protein mRNA detected using synthetic oligonucleotide probes.

Synthetic oligonucleotide probes were designed to detect the alpha-subunits of the guanine-nucleotide-regulatory proteins (G-proteins) Gi-1, Gi-2, Gi-3 and Gs (Gi is inhibitory and Gs is stimulatory). Each probe detected a single major mRNA species in Northern blots of RNA extracted from a variety of tissues. A probe was designed to identify the two forms of G-protein beta-subunits, beta 1 and beta 2. This probe hybridised with a single 1.8-kb transcript (beta 2) in RNA from all tissues studied except for brain, where a less-abundant 3.4-kb transcript (beta 1) was also detected. These probes were used to assess whether the induction of diabetes, using streptozotocin, altered the levels of mRNA coding for specific G-protein components. In hepatocytes, diabetes caused a significant reduction in the number of transcripts coding for alpha-Gs, alpha-Gi-2 and alpha-Gi-3; mRNA for alpha-Gi-1 was undectable. In adipocytes, diabetes increased dramatically the mRNA coding for alpha-Gi-1 and alpha-Gi-3, whilst no significant changes occurred in the fractions coding for alpha-Gi-2 and alpha-Gs. No significant changes in the mRNA coding for G-protein alpha-subunits were observed in either brain, heart, skeletal muscle or kidney. Diabetes did not cause any significant changes in the mRNA coding for beta 2 in any tissue or cell population studied. Such results on the relative levels of mRNA encoding G-protein components was obtained by comparing equal amounts of total RNA from tissues of control and diabetic animals. G-protein mRNA levels were expressed relative to ribosomal 28S RNA levels and, in some instances, relative to transcripts for a structural protein called CHO-B. The total cellular levels of both RNA and DNA were assessed in the various tissues and cells studied. Major falls in RNA levels/cell appeared to occur in hepatocytes and to a lesser extent in adipocytes and skeletal muscle. Thus major reductions in G-protein transcripts occurred in hepatocytes. The detected changes in G-protein mRNA are discussed in relation to the available evidence on G-protein expression. We suggest that diabetes causes tissue-specific changes in the levels of mRNA for particular G-protein species; this may have consequences for the functioning of cellular signal-transduction mechanisms in the affected tissues.

Diabetes-induced alterations in the expression, functioning and phosphorylation state of the inhibitory guanine nucleotide regulatory protein Gi-2 in hepatocytes.

Levels of the G-protein alpha-subunits alpha-Gi-2, alpha-Gi-3 and the 42 kDa, form of alpha-Gs were markedly decreased in hepatocyte membranes from streptozotocin-diabetic animals as compared with normals. In contrast, no detectable changes in alpha-Gi subunits were seen in liver plasma membranes of streptozotocin-diabetic animals, although levels of the 45 kDa form of Gs were increased. G-protein beta subunits in plasma membranes were unaffected by diabetes induction. Analysis of whole-liver RNA indicated that the induction of diabetes had little effect on transcript levels of Gi-3, caused an increase in Gs transcripts and decreased transcript number for Gi-2, albeit to a much lesser extent than was observed upon analysis of hepatocyte RNA. In both hepatocyte and liver plasma membranes, immunoblot analysis showed that levels of the catalytic unit of adenylate cyclase were increased upon induction of diabetes. Under basal conditions, alpha-Gi-2 from hepatocytes of diabetic animals was found to be both phosphorylated to a greater extent than alpha-Gi-2 isolated from hepatocytes of normal animals, and furthermore was resistant to any further phosphorylation upon challenge of hepatocytes with angiotensin, vasopressin or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. Treatment of isolated plasma membranes from normal, but not diabetic, animals with purified protein kinase C caused the phosphorylation of alpha-Gi-2. Treatment of membranes from diabetic animals with alkaline phosphatase caused the dephosphorylation of alpha-Gi-2 and rendered it susceptible to subsequent phosphorylation with protein kinase C. Low concentrations of the non-hydrolysable GTP analogue guanylyl 5'-imidodiphosphate inhibited adenylate cyclase activity in both hepatocyte and liver plasma membranes from normal, but not diabetic, animals.

Guanine-nucleotide-binding proteins expressed in rat white adipose tissue. Identification of both mRNAs and proteins corresponding to Gi1, Gi2 and Gi3.

Considerable debate has focused on the molecular identity of the guanine-nucleotide-binding proteins (G-proteins) in adipose tissue which can be detected following pertussis-toxin-catalysed ADP-ribosylation [Rapiejko, Northup, Evans, Brown & Malbon (1986) Biochem. J. 240, 35-40; Hinsch, Rosenthal, Spicher, Binder, Gausepohl, Frank, Schultz & Joost (1988) FEBS Lett. 238, 191-196]. We have used a panel of selective anti-peptide antisera which are able to discriminate between the different pertussis-toxin-sensitive G-proteins to assess which of these are expressed in rat adipose tissue. We demonstrate that plasma membranes of rat white adipocytes contain alpha subunits corresponding to each of Gi1, Gi2 and Gi3. Furthermore, using synthetic oligonucleotides complimentary to unique regions of each of the three polypeptides, we demonstrate that the mRNAs for the three G-protein alpha subunits can also be detected in adipose tissue.

Characterization of the receptor for cholera toxin and Escherichia coli heat-labile toxin in rabbit intestinal brush borders.

125I-labelled heat-labile toxin (from Escherichia coli) and 125I-labelled cholera toxin bound to immobilized ganglioside GM1 and Balb/c 3T3 cell membranes with identical specificities, i.e. each toxin inhibited binding of the other. Binding of both toxins to Balb/c 3T3 cell membranes was saturable, with 50% of maximal binding occurring at 0.3 nM for cholera toxin and 1.1 nM for heat-labile toxin, and the number of sites for each toxin was similar. The results suggest that both toxins recognize the same receptor, namely ganglioside GM1. In contrast, binding of 125I-heat-labile toxin to rabbit intestinal brush borders at 0 degree C was not inhibited by cholera toxin, although heat-labile toxin inhibited 125I-cholera toxin binding. In addition, there were 3-10-fold more binding sites for heat-labile toxin than for cholera toxin. At 37 degrees C cholera toxin, but more particularly its B-subunit, did significantly inhibit 125I-heat-labile toxin binding. Binding of 125I-cholera toxin was saturable, with 50% maximal of binding occurring at 1-2 nM, and was quantitatively inhibited by 10(-8) M unlabelled toxin or B-subunit. By contrast, binding of 125I-heat-labile toxin was non-saturable (up to 5 nM), and 2 X 10(-7) M unlabelled B-subunit was required to quantitatively inhibit binding. Neuraminidase treatment of brush borders increased 125I-cholera toxin but not heat-labile toxin binding. Extensive digestion of membranes with Streptomyces griseus proteinase or papain did not decrease the binding of either toxin. The additional binding sites for heat-labile toxin are not gangliosides. Thin-layer chromatograms of gangliosides which were overlayed with 125I-labelled toxins showed that binding of both toxins was largely restricted to ganglioside GM1. However, 125I-heat-labile toxin was able to bind to brush-border galactoproteins resolved by SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose.

Variants of BALB/c 3T3 cells lacking complex gangliosides retain a fibronectin matrix and spread normally on fibronectin-coated substrates.

Evidence has accumulated that di- and trisialogangliosides are involved in the interaction of cells with fibronectin. We have therefore tested the ability of variants of BALB/c 3T3 deficient in such gangliosides to organize a fibronectin matrix and to spread on fibronectin-coated substrates. Whereas BALB/c 3T3 cells contained gangliosides GM3, GM1, and GD1a, direct chemical analysis showed that five out of six variants isolated contained no detectable GD1a. By the overlaying of thin layer chromatograms of cellular gangliosides with 125I-cholera toxin, these variants were also found to lack ganglioside GM1. In contrast, the sialogalactoprotein profile of these cells, analyzed using an 125I-ricin/SDS polyacrylamide gel overlay technique, was similar to that of the parent cell line. All variants organized an extensive fibronectin matrix comparable to that of BALB/c 3T3, as shown using either immunofluorescence or lactoperoxidase-catalyzed iodination. The variants could also spread on fibronectin-coated substrates and adopt a morphology similar to that of BALB/c 3T3 cells, with little or no difference in the concentration of fibronectin required for 50% cell spreading. Cell spreading of the variants was accompanied by the formation of focal contacts and microfilament bundles, in a manner closely resembling that seen with BALB/c 3T3 cells. Treatment of BALB/c 3T3 cells with neuraminidase, which converts much of the cellular GD1a to GM1, did not affect cell spreading on fibronectin. The results clearly demonstrate that complex gangliosides are not essential for retention of a fibronectin matrix or for spreading on fibronectin-coated substrates.

Coping with old age in a Philippine emigrant community.

Because of the predominantly male migration to Hawaii in the 1920s and 1930s, there are many women in this Philippine community who have never married. These spinsters become dependent on nieces or nephews for a livelihood. Today, both men and women are going abroad, and grandparents often care for grandchildren, not only to provide a service for their emigrant children, but also to ensure the flow of remittances. Comparatively wealthy single men who return from Hawaii for their retirement marry young women and begin families. In addition to providing spouses to care for them in their old age, May-December marriages incorporate the retirees into the social and economic structure of the community. ka]Key Words kb]retirees kb]spinsters kb]emigrant communities kb]May-December marriages kb]remittances kb]Hawaii.

Isolation of variants of BALB/c 3T3 cells defective in complex ganglioside biosynthesis.

In an attempt to clarify the relationship between altered glycosphingolipid metabolism and other aspects of the transformed phenotype, we have isolated variants of BALB/c 3T3 cells (clone A31) which are defective in synthesis of the more complex gangliosides (GM2, GM1 and GD1a). The selection protocol was based on the specificity of cholera toxin for ganglioside GM1, and the ability to lyse cells which bound toxin using anti-toxin and complement. Following treatment of cells with ethane methane sulfonate (EMS), populations resistant to lysis were obtained after 5-6 rounds of selection, despite a low and rather variable killing efficiency (75-95%). Five out of six clones isolated from such populations showed reduced toxin-binding capacity and loss of gangliosides more complex than GM3, as determined by metabolic labelling with [1-14C] palmitate. An identical phenotype was displayed by a variant isolated from a non-mutagenized population of cells. The phenotype remained stable for several months in culture and for over at least 40 cell doublings. Ganglioside nomenclature is according to Svennerholm [24].